Recently, animal cell culture technology has been remarkably developed, and also research and development targeting animal cells have been carried out over a variety of wide-scale fields. At the first stage of development, animal cells as a target have been used for making products of the cells themselves or making products of materials produced by the cells. Currently, cells and their surface proteins are analyzed to design useful medicines. Furthermore, treatment by growing patients' cells in vitro or enhancing the function of the cells and then returning the cells to the patients' bodies being put into practice. At present, the technology for culturing animal cells is one field to which many researchers pay attention.
Many animal cells including human cells are anchorage-dependent. That is, in order to culture animal cells in vitro, the cells must be once attached to something. Against such background, many researchers have studied or developed substrate surfaces that are more desirable for cells before, but all these technologies are involved in those during cell culture. Cultured anchorage-dependent cells produce adhesive proteins by themselves when they adhere to something. The adhesive proteins must therefore be destroyed usually by enzyme treatment in order to detach the cells in conventional techniques. On such occasion, cell surface proteins that are produced by the cells during the culture and are inherent to various types of cells are also simultaneously destroyed. This severe problem, however, has no actual countermeasure and has not been investigated very often. It is believed that this problem on the collection of cells should be solved for breakthrough in research and development using animal cells in future.
Against such background, JP-A-Hei-2-211865 describes a new method of cell culture including culturing cells on a cell culture support having a substrate surface coated with a polymer having an upper or lower critical solution temperature to water of 0 to 80° C. at a temperature lower than the upper critical solution temperature or not lower than the lower critical solution temperature and then detaching the cultured cells without enzyme treatment by changing the culturing temperature to a temperature of not lower than the upper critical solution temperature or lower than the lower critical solution temperature. JP-A-Hei-5-192138 describes a method of culturing skin cells using this temperature-responsive cell culture substrate at a temperature lower than the upper critical solution temperature or not lower than the lower critical solution temperature and then detaching the cultured skin cells with low damage by changing the culturing temperature to a temperature of not lower than the upper critical solution temperature or lower than the lower critical solution temperature. In addition, Japanese Patent Application No. 2007-105311 describes a method of repairing surface protein of the cultured cells using this temperature-responsive cell culture substrate. The use of the temperature-responsive cell culture substrate contributes to further development of conventional culturing technology.
The conventional culturing technology has been developed in diverse new ways using the temperature-responsive cell culture substrate. However, in this technology, the entire surface of the cell culture substrate is uniformly coated with a temperature-responsive polymer, and the size of the resulting cell sheet always depends on the size of culture substrate. In addition, the cell sheet has a circular shape because the shapes of the culture surfaces of existing culture vessels are usually circular. Accordingly, a disadvantage of the shape is that the cell sheets cannot efficiently arranged on an affected area when they are grafted onto the affected area. Consequently, there is a demand for more precisely designing the temperature-responsive portion of the culture substrate surface.
Regarding the coating state of this temperature-responsive polymer, Japanese Patent No. 3441530 discloses a surface including a region having a specific affinity and a stimulation-responsive polymer region locally in the culturing surface or in a patterned form. However, this method to produce the substrate surface requires at least two steps for forming the respective regions on the same substrate surface and, therefore, is not efficient or simple. Similarly, Japanese Patent No. 3641301 discloses a surface including a region having a specific affinity and a stimulation-responsive polymer region locally in the culturing surface or in a patterned form. In also this technology, the production process of the substrate surface is not simple due to the reasons described above, and there is a demand for developing a new method.